A double-stranded RNA (dsRNA)-activated protein kinase is one of three enzymes known to be induced in animal cells following interferon treatment. The antiviral effects of interferon, and possibly the anticellular properties of interferon may be partly explained by the action of this enzyme in phosphorylating, and thus inactivating eucaryotic protein synthesis initiation factor (eIF-2). Inhibition of eIF-2 action is not completely understood nor is the mechanism of action of the kinase, or even whether eIF-2 is phosphorylated in vivo in virus infected interferon-treated cells. A deeper understanding of dsRNA-kinase action requires purification of the enzyme. This should reveal whether the kinase can use substrates other than eIF-2 and histones, whether accessory proteins are necessary for activation of the enzyme by dsRNA, and whether a phosphorylated protein (72,000 Mr) is a kinase subunit, a regulatory subunit or merely another substrate. The enzyme will be isolated from cultures of HeLa cells treated with human fibroblast interferon following experiments designed to maximize the yield of dsRNA-activated eIF-2 kinase. Assay conditions and methods for detection of the phosphorylated products will be optimized prior to fractionation of the enzyme. The purified enzyme will be used to prepare monoclonal antibodies to the kinase. These should prove to be useful in analyzing the action of the enzyme in interacting with a number of other proteins, substrates and dsRNA. Monoclonal antibodies will also be used to probe for in vivo phosphorylated proteins following EMC virus infection of interferon treated HeLa cells and to study functions of phosphorylated eIF-2 in protein synthesis.